Redox balances in the metabolism of sugars by yeasts

Abstract The central role of the redox couples NAD⁺/NADH and NADP⁺/NADPH in the metabolism of sugars by yeasts is discussed in relation to energy metabolism and product formation. Besides their physical compartmentation in cytosol and mitochondria, the two coenzyme systems are separated by chemical compartmentation as a consequence of the absence of transhydrogenase activity. This has considerable consequences for the redox balances of both coenzyme systems and hence for sugar metabolism in yeasts.
As examples, the competition between respiration and fermentation of glucose, the Crabtree effect, the Custers effect, adaptation to anaerobiosis, the activities of the hexose monophosphate pathway, and the fermentation of xylose in yeast are discussed.     

Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [ND_in(NADPH)] in the oxidation of strictly NAD⁺-linked substrates by pea ( Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 μ M diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of ND_in(NADPH) during glycine oxidation (measured both as O₂ uptake and as CO₂ release) was 40–50 nmol mg⁻¹ protein min⁻¹. No significant activity of ND_in(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD⁺-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 μ M , DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, ND_in(NADPH), and ND_in(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD⁺ and NADP⁺, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.     

Correlations between the ascorbate-glutathione pathway and effectiveness in legume root nodules

Legume root nodules use the ascorbate-glutathione pathway to remove harmful H₂O₂. In the present study. effective and ineffective nodules from soybean and alfalfa were compared with regard to this pathway. Effective nodules had higher activity of all 4 enzymes (ascorbate peroxidase, EC 1. 11. 1. 11: monodehydroascorbate reductase, EC 1. 6. 5. 4: dehydroascorbate reductase, EC 1. 8. 5. 1: and glutathione reductase, EC 1. 6. 4. 2). The concentration of thiol tripeptides (primarily homoglutathione) was about 1 m M in effective nodules – a level 3–4-fold higher than in ineffective nodules. Effective nodules contained higher levels of NAD⁺. NADP⁺ and NADPH. but not of NADH or ascorbate. The increased capacity for peroxide scavenging in effective nodules as compared to ineffective nodules emphasizes the important protective role that this pathway may play in processes related to nitrogen fixation.     

NADP+-malate dehydrogenase in C3-plants: Regulation and role of a light-activated enzyme

The thioredoxin-dependent light/dark modulation system of the chloroplast is described as a prerequisite enabling the flexible control of fluxes through the various parts of the CO₂-fixation pathway. Both the rapid turnover of the reduced thiol-containing form of the respective target enzyme, and the metabolite effect upon the reductive enzyme modulation, allow rapid adjustment of the amount of active species to the actual requirements. The structural basis of the regulation of chloroplast NADP⁺-malate dehydrogenase (EC 1.1.1.82) is described in more detail. The modulable plastid enzyme is characterized by two sequence extensions not present in any other known NADP⁺- and/or NAD⁺-specific malate dehydrogenase. The NADP⁺-malate dehydrogenase of C₃-plants is part of the "malate valve", which catalyzes the export of reducing equivalents in the form of malate from the chloroplast only when the NADPH to NADP⁺ ratio is high, thus poising the NADPH to ATP ratio required for optimal carbon reduction in the light. The mode of regulation of other light/dark modulated enzymes is discussed.     

THE EFFECT OF MODERATE AND MARKED HYPERCAPNIA UPON THE ENERGY STATE AND UPON THE CYTOPLASMIC NADH/NAD+ RATIO OF THE RAT BRAIN

Abstract— The energy state of brain tissue was evaluated from the tissue concentrations of ATP, ADP and AMP and the cytoplasmic NADH/NAD⁺ ratio from the tissue, CSF and blood concentrations of lactate and pyruvate, and from the intracellular pH', in rats exposed to carbon dioxide concentrations of 640 per cent. The hypercapnia had no significant effect on the energy state of the tissue. Hypercapnia of increasing severity gave rise to a progressive decrease in the pyruvate concentration; the lactate concentration fell at low CO₂ concentrations, but no further decrease was observed at CO₂ concentrations greater than 20 per cent. There was a progressive rise in the intracellular lactate/pyruvate ratio at increasing CO₂ concentrations, corresponding to the fall in intracellular pH, i.e. the calculated NADH/NAD⁺ ratios remained normal. It is therefore concluded that hypercapnia does not affect the cytoplasmic redox state.     

Vacuolating cytotoxic activity of Pasteurella multocida causing haemorrhagic septicaemia in buffalo and cattle

Abstract Sequence data had indicated that cyanobacteria might possess a bidirectional hydrogenase with properties similar to the soluble enzymes from Alcaligenes eutrophus, Nocardia opaca and Desulfovibrio fructosovorans . The present study shows that extracts from the cyanobacterium Anacystis nidulans catalyse NAD(P)H-dependent H₂ evolution with low but significant activity and uptake of the gas with NAD(P)⁺ as the electron acceptor. NAD⁺ is the preferred electron acceptor and NADH the preferred donor compared to NADP⁺ and NADPH, respectively. Activity levels of this NAD(P)⁺dependent, bidirectional hydrogenase are too low to support chemoautotrophic growth in A. nidulans .     

ATPase activity of isolated plasma membrane vesicles of leaves of Elodea as affected by thiol reagents and NADH/NAD+ ratio

The goal of this study was to test the hypothesis that the plasma membrane-bound ATPase activity is influenced by the redox poise of the cytoplasm. Purified plasma membrane vesicles from leaves of Elodea canadensis Michx. and E. nuttallii (Planch.) St. John were isolated using an aqueous polymer two-phase batch procedure. The distribution of marker enzyme activities confirmed the plasma membrane origin of the vesicles. The vesicles exhibited NADH-ferricyanide reductase activity, indicating the presence of a redox chain in the plasma membrane. The K⁺, Mg²⁺-ATPase activity associated with these vesicles was inhibited by the sulfhydryl reagents N-ethylmaleimide and glutathione (GSSG). Furthermore the activity was inhibited by NAD⁺. This inhibition by NAD⁺ was relieved by increasing the NADH/NAD⁺ ratio. The possibility that the ATPase activity is regulated by the cytoplasmic NAD(P)H/ NAD(P)⁺ ratio is discussed, as well as the role of a plasma membrane-bound redox chain.     

THE ENERGETICS OF EXTRACELLULAR FE(III) REDUCTION BY IRON-LIMITED CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

Fe-limited cells of the green alga Chlamydomonas reinhardtii (Fe-limited growth rate = 0.3 d⁻¹) reduced extracellular Fe(III) to Fe(II) when Fe(III) was supplied as ferricyanide or Fe(III)-EDTA; Fe(III) reduction was stimulated by light. In both darkness and during photosynthesis, ferricyanide reduction was accompanied by a decrease in cellular NADPH levels, with a concomitant increase in NADP⁺. NADH and NAD⁺ levels were not measurably altered during ferricyanide reduction. Furthermore, cellular hexose monophosphate levels declined and 6-phosphogluconate levels increased during ferricyanide reduction. Levels of most glycolytic and tricarboxylic acid cycle intermediates were mostly unaltered. Ferricyanide reduction was also associated with a decrease in cellular ATP levels, a concomitant increase in ADP and AMP, and increased extracellular acidification. The acidification was sensitive to inhibition by the H⁺-ATPase inhibitor N,N' -dicyclohexylcarbodiimide (DCCD). We conclude that the oxidative pentose phosphate pathway provides reducing equivalents for Fe(III) reduction in darkness and also contributes reducing equivalents to Fe(III) reduction during photosynthesis. The decline in ATP was likely due to activation of the plasma membrane H⁺-ATPase during ferricyanide reduction and was not directly associated with provision of reducing equivalents.     

Characteristics of the reverse reaction of NADP+-isocitrate dehydrogenase from castor bean seeds

The reductive carboxylation of α-ketoglutarate by purified NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) from maturing castor bean seeds ( Ricinus communis L. ) has been characterized. The optimum pH for the reaction was 6.5, whereas pH 8.5 was optimum for oxidation of isocitrate (forward reaction). The enzyme utilized NADH as well as NADPH as the reducing agent in the reverse reaction, but only NADP⁺ in the forward reaction. The Km values for NADPH and NADH were 0.044 and 2.8 m M respectively, and for α-ketoglutarate and HCO₃ 4.1 and 3.7 m M. The enzyme was activated by various cations including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺ and Co²⁺. Km values for Mg²⁺ Mn²⁺, Co²⁺ and Zn²⁺ were 12, 34, 37 and 49μ M respectively.     

Changes in the level of oxidized and reduced pyridine nucleotides during cold acclimation of winter rape plants

Prolonged cold (2°C) treatment of winter rape plants ( Brassica napus L. var. oleifera L. cv. Górczański) markedly modified the pattern of leaf growth and brought about changes in the level of pyridine nucleotides already during the first few days of treatment. The NAD⁺, NADP⁺ and NADPH levels markedly increased but there was practically no effect on the NADH level. Changes in the respective nucleotide levels were reflected by changes in anabolic and catabolic reduction charges. The former increased by 70%, whereas the latter decreased by 44%. Alterations in pyridine nucleotide levels and the reduction charges are discussed in terms of possible mechanisms involved, as well as in terms of their role in plant adaptation to cold.     

Properties of the glyceraldehyde-3-P dehydrogenase in heterocysts and vegetative cells of cyanobacteria

Abstract Glyceraldehyde-3-P dehydrogenase (GAPDH) in heterocysts and vegetative cells of 3 N₂-fixing cyanobacteria was found to utilize both NAD⁺ and NADP⁺. The enzyme activity was enhanced by thiols (glutathione, reduced lipoic acid and dithiothreitol). GAPDH of the 3 cyanobacterial species was not activated by thioredoxin. Heterocysts have now been shown to possess all the enzymes of glycolysis and the tricarboxylic acid cycle to convert glyceraldehyde-3-phosphate (GAP) to oxoglutarate and glutamate. The GAPDH reaction is a major source for the generation of NADH, which is oxidized by a thylakoid-bound NADH:plastoquinone oxidoreductase in heterocysts.     

Energy Metabolism in Rat Brain Synaptosomes from Nembutal-Anesthetized and Nonanesthetized Animals

Abstract: Cellular energetic parameters including the intramitochondrial and cytosolic [NAD⁺]/[NADH] ratios, the cellular [ATP]/[ADP][P_i and [creatine phosphate]/[creatine] ratios, the concentration of cytochrome c and its redox state and the respiratory rate were studied in suspensions of rat brain synapto-somes isolated from nembutal-anesthetized and nonanesthetized animals. The ratio of [3-hydroxybutyrate] to [acetoacetate] was 2.0 in synaptosomes isolated from nonanesthetized rats and 5.55 in those from anesthetized animals. The [lactate]/[pyruvate] ratio was 3.8 in the former and 10.9 in the latter preparation. The [ATP]/[ADP][P_i] was 3838 M⁻¹ in the synaptosomes from anesthetized rats and 840 M⁻¹ in those from nonanesthetized animals and the [creatine phosphate]/[creatine] ratios were 0.79 and 0.39, respectively. Cytochrome c was about 15% reduced in both preparations; however, the mitochon-drial cytochrome concentration was almost twofold higher in the synaptosomes from nonanesthetized animals. Calculations of the free energy relationships between the mitochondrial redox reactions and ATP synthesis showed that in synaptosomes isolated from the brains of nembutal-anesthetized rats the first two sites of oxidative phosphorylation were at near-equilibrium, in agreement with observations for intact cells and tissues. The energetic parameters for synaptosomes from anesthetized rats are very similar to the values for intact whole brain, whereas those for synaptosomes from nonanesthetized rats are lower and suggest that nembutal anesthesia protects against some irreversible damage to the synaptosome during isolation. It is concluded that synaptosomes isolated from brains of nembutal-anesthetized rats can be used as a convenient model system for studies of neuronal metabolism.     

Purification and properties of a novel polyol dehydrogenase of bacterial origin

Abstract A bacterium, as yet unidentified, has been isolated from floor dust by direct selection on minimal agar using l -glucitol ( d -gulitol) as the sole carbon energy source. The bacterium possesses a constitutive enzyme which catalyzes the reaction: l -glucitol + NAD⁺→ d -sorbose + NADH + H⁺. A new species of enzyme has been induced by l -arabinitol or ribitol, but not l - or d -glucitol, and the induction is only partially counteracted by the glucose-repression effect. The constitutive enzyme was purified by fractionation on Sephadex G-200 gel and chromatography on DEAE Biogel A. The enzyme required NAD⁺, but not NADP⁺, as a cofactor. It oxidizes also ribitol, xylitol and l -arabinitol, but not d -arabinitol, lactitol or a variety of other commercially available alditols. The enzyme is not inhibited by 10 mM sodium azide but is totally inhibited by 0.1 mM potassium ferricyanide.     

Expression of Azotobacter vinelandii soluble transhydrogenase perturbs xylose reductase-mediated conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae

Pyridine nucleotide transhydrogenase is a metabolic enzyme transferring the reducing equivalent between two nucleotide acceptors such as NAD⁺ and NADP⁺ for balancing the intracellular redox potential. Soluble transhydrogenase (STH) of Azotobacter vinelandii was expressed in a recombinant Saccharomyces cerevisiae strain harboring the Pichia stipitis xylose reductase (XR) gene to study effects of redox potential change on cell growth and sugar metabolism including xylitol and ethanol formation. Remarkable changes were not observed by expression of the STH gene in batch cultures. However, expression of STH accelerated the formation of ethanol in glucose-limited fed-batch cultures, but reduced xylitol productivity to 71% compared with its counterpart strain expressing xylose reductase gene alone. The experimental results suggested that A. vinelandii STH directed the reaction toward the formation of NADH and NADP⁺ from NAD⁺ and NADPH, which concomitantly reduced the availability of NADPH for xylose conversion to xylitol catalyzed by NADPH-preferable xylose reductase in the recombinant S. cerevisiae.     

In vitro and in vivo regulation of chJoroplast glyceraldehyde-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. III. The molecular basis of the aggregation phenomenon: chloroplast glyceraldehyde-3-phosphate dehydrogenase as an ambiquitous enzyme

The nature of the aggregated form of chloroplast glyceraldehyde-3-phosphate dehydrogenase isozymes (GPD, EC 1.2.1.13) from Chenopodium rubrum leaves was investigated. After disaggregation of the isozymes in NADP ⁺ buffer, and resuspension of the disaggregated isozymes in NAD⁺ buffer, complete reaggregation could only be achieved by remixing the enzyme with a high molecular weight fraction, from which the isozymes had dissociated during the NADP⁺ filtration. After separation of the isozymes by inverse ammonium sulphate gradient solubilization, spontaneous extensive reaggregation of each isozyme was observed in NAD⁺ buffer. The high molecular weight material consisted of ribonucleoprotein, and RNase treatment impaired its ability to promote reaggregation of chloroplast GPD. It is proposed that pyridine nucleotide-controlled aggregation and binding to ribonucleoprotein in vitro are artifacts which reflect an in situ binding to cellular components. Since uncontrolled NAD⁺-linked activities of the bifunctional isozymes in the chloroplast would lead to an equalization of the NAD ⁺ and NADP ⁺ redox couples, it is suggested that the reversible binding of the isozymes forms the basis of a regulatory system in vivo.     

MECHANISMS ACTIVATING GLYCOLYSIS IN THE BRAIN IN ARTERIAL HYPOXIA

Abstract— In order to study regulatory steps responsible for the activation of anaerobic glycolysis in the brain during hypoxia, cerebral concentrations of carbohydrate substrates and organic phosphates were measured in rats after reduction of the arterial P_O2 to 23-25 mm Hg for 2, 5 and 15 min. The results demonstrated a progressive accumulation of lactate as well as of pyruvate and malate in the absence of changes in ATP, A DP, AMP, citrate and ammonia. The pattern of substrate changes obtained indicate that hypoxia is accompanied by activation of pyruvate kinase and of hexokinase, but not of phosphofructokinase. There was a progressive fall in intracellular pH and a moderate increase in the calculated cytoplasmic NADH/NAD⁺ ratio. The changes in pyruvate and in the NADH/NAD⁺ ratio may be responsible for the observed increase in the malate concentration.     

Energy conservation in Nitrobacter

Abstract The generation of ATP and NADH in total cells of Nitrobacter was measured under aerobic and anaerobic conditions. NADH synthesis was driven by an ATP independent reaction with nitrite or nitric oxide as electron donors. The rate of NADH formation was about 200 times higher, if nitric oxide instead of nitrite served as electron donor. Approximately 2 mol nitric oxide were needed for reduction of 1 mol NAD⁺. Nitrite caused an end-product inhibition of the nitric oxide induced NADH synthesis. ATP was synthesized by NADH oxidation with oxygen and nitrate as terminal electron acceptors.     

FREEZE-BLOWING: A NEW TECHNIQUE FOR THE STUDY OF BRAIN IN VIVO

Abstract— A new apparatus is described which removes and freezes brains of conscious rats more rapidly than was heretofore possible. The apparatus consists of two probes which are driven simultaneously into the cranial vault of the rat immobilized in a specially constructed restraining cage. When in position, air under pressure enters through one probe and blows the supratentorial portion of the brain tissue (situated between the olfactory bulbs and the superior colliculi) out the other probe and into a thin chamber previously cooled in liquid N₂. This method stops brain tissue metabolism more rapidly than the previously-described methods of microwave irradiation, decapitation into liquid N₂, or whole-animal immersion into liquid N₂, as evidenced by the measurement of labile metabolites and redox states. Thus, samples of freeze-blown brain had higher levels of a-oxoglutarate, creatine phosphate, pyruvate, glucose and glucose-6-phosphate and lower levels of lactate, malate and AMP than brain tissue obtained by the other methods. The free cytoplasmic [NAD⁺]/[NADH₂], [NADP⁺]/[NADPH₂] and [ATP]/[ADP] [HPO₄^2-] ratios were higher in freeze-blown samples. These data indicate that more extensive anoxic metabolism occurred when methods other than freeze-blowing were used. We conclude that the levels of metabolites measured in brain obtained with the freeze-blowing technique more closely resemble those which occur in vivo.     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

The oxidation of cytosolic NAD(P)H by external NAD(P)H dehydrogenases in the respiratory chain of plant mitochondria

The respiratory chain of plant mitochondria differs from that in mammalian mitochondria by containing several rotenone-insensitive NAD(P)H dehydrogenases. Two of these are located on the outer, cytosolic surface of the inner membrane. One is specific for NADH, the other for NADPH. Only the latter is inhibited by diphenyleneiodonium (DPI). Both of these enzymes are normally dependent upon Ca²⁺ for activity and this constitutes a potentially important mechanism by which the cell can regulate the oxidation of cytosolic NAD(P)H via the concentration of free Ca²⁺. This and other potential regulatory mechanisms such as the substrate concentration and polyamines are discussed.